(a–d) Differential interference contrast (DIC) fluorescence overlay of live human MSCs expressing fluorescent proteins that target mitochondria (green) and phagosomes (red) shows mitochondria being loaded into phagosomes (arrows), which are then shuttled to the plasma membrane for extrusion (also see ). (e–h) Inset shows a representative macrophage interacting with a human MSC. This interaction is shown as a time sequence (5 min intervals) in the lower images and in . The inset demarcates the area in the human MSC plasma membrane where the membrane blebs outwards and accumulates vesicles. Macrophages nibble the surface of human MSCs and uptake mitochondrial laden phagosomes from blebs budding (arrows) from the plasma membrane of the human MSCs. Scale bars, 10 μ.

(a) Top panel is DIC fluorescent overlay at time 0 of primary human MSCs infected with Organelle Lights to label mitochondria (green) and co-cultured with mouse (RAW 264.7) macrophages. Lower panels, time sequence at 45 min intervals showing transfer of green-labelled mitochondria from the a MSC to a macrophage (red arrow, see for transfer of mitochondria in filamentous form, and in which GFP signal is compensated to allow the tracking of the transferred mitochondria into macrophages). (b) Left panel, photomicrograph of FACS-sorted mouse macrophages that were co-cultured with mitochondria-labelled (RFP) human MSCs clearly show retention of RFP label. Right panel, electrophoretic pattern of human COX I PCR product treated with or without Bfa1 after amplification from the indicated cell sources. (c) MSC-derived exosomes and MVs express the Bfa1-sensitive 228-bp COX I mtDNA PCR product detected in human MSCs (b). (d) Left panel, electrophoretic pattern of Bfa1-digested human COX1 PCR product amplified from mouse lung DNA isolated 14 days after the intravenous administration of human MSCs, human MSC-derived MVs or exosomes. Right panel, human GAPDH and human COX1 relative expression levels quantified by RT–PCR in mouse lung (3–28 days) after a single (intratracheal (IT) or intravenous (IV)) injection of human MSCs, human MSC-derived exosomes or human fibroblasts. *P<0.001, #P<0.001 by ANOVA compared with untreated mouse lung. Plotted values (mean±s.e.m.) are from experiments repeated four times. Scale bars, 20 μ.

(a) Mitochondrial respiration of human macrophages, human MSCs or human fibroblasts was measured as OCR using the XF technology. Macrophages were co-cultured with or without human MSCs or fibroblasts (1:10 ratio) or treated with human MSC-derived exosomes (40 μg per protein) in the presence or absence of Oligomycin A and FCCP to differentiate ATP-linked respiration from the proton leak. Plotted data (mean±s.e.m.) were performed using six replicates per sample and repeated three times. (b) Pseudocoloured photomicrographs (0–240 min) of MitoSOX Red-stained macrophages that were non-stimulated (upper panel), or treated with silica (20 μg cm−2, lower panel) or silica plus human MSC-derived exosomes (added 10 min after silica, middle panel). Scale bars, 50 μ. (c) Time course of MitoSOX Red emission by human macrophages treated as in b. Figure is representative of five exposures (nine stages positions per test and 6 cells per stage). (d) OCR as in a of silica-exposed macrophages treated with or without human MSCs, human MSC-derived exosomes or human fibroblasts. Plotted values (mean±s.e.m.) are from experiments repeated three times, *P<0.05 as compared to control, #P<0.05 as compared to silica treated macrophages, as determined by Student's t-test.

Mitochondrial transfer from human MSCs is followed by fusion inside macrophages.

Human MSCs and human macrophages (1 × 105) were infected separately with Organelle Lights to label human MSC mitochondria (red) and macrophage mitochondria (green). Twenty-four hours following infection, macrophages were harvested and co-incubated with the human MSCs for 2 h. Images were collected using an inverted Nikon TiE fluorescent microscope equipped with a × 60 oil immersion optic and NIS Elements Software. Organelle Lights were excited using a Lumencor diode-pumped light engine and detected using an ORCA-Flash4.0 sCMOS camera. (a,b) DIC images of two separate fields within the same dish. (c) A zoomed image of the outlined section within b (scale bars, 20 μ). The fluorescence-based images for each field appear in the panels below the DIC images, with d–f showing macrophage mitochondria (green); g–i showing human MSC mitochondria (red); and j–l showing the overlay with yellow indicative of colocalization of human MSC and macrophage mitochondria. Not every macrophage was shown to take up human MSC mitochondria (a,d,g, j).